Microbial polysaccharide and process



A nl 18, 1967 cADMus ETAL 3,314,801

MICROBIAL POLYSACCHARIDEAND PROCESS Original Filed om 24, 1963 4 Sheets-Sheet 1 [27000 POLYMER INCULTURE L2 (D POLYMER CONCENTRATION (q/looml) VISCOSITY (CENTIPOISE) ENZYME- HYDROLYZED CASEIN /looml) FIG! INVENTORS ATTORNEYS Apnl 18, 1967 M. c. CADMUS ETAL 3,3

MICROBIAL POLYS'ACCHARIDE AND PROCESS Original Filed Oct. 24. 1963 4 Sheets-Sheet 5 VISCOSIW(CENTIPO1SE x10 20 no hem IO V no KCI no hen? POLYMER CONCENTRATION (q loom!) H63 INVENTURS MARTIN C.CADMUS RALPH EANDERSON ATTORNFYS United States Patent fifice 3,314,801 MICROBIAL POLYSACCHARIDE AND PROCESS Martin C. Cadmus and Ralph F. Anderson, Peoria, IlL, assignors to the United States of America as represented by the Secretary of Agriculture Original application Oct. 24, 1963, Ser. No. 318,769, new Patent No. 3,228,855, dated Jan. 11, 1966. Divided and this application Aug. 17, I964, Ser. No. 399,117 1 Claim. (Cl. 99-439) A nonexclusive, irrevocable, royalty-free license in the invention hereindescribed, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This application is a division of application bearing Ser. No. 318,769, filed Oct. 24, 1963, now Patent No. 3,228,855, granted Jan. 11, 1966.

This invention relates to a novel microbial polysaccharide, aqueous solutions of which novel polysaccharide polymer display unique viscosity characteristics that make the edible polymer particularly advantageous as a thickening agent broadly and as an additive for oil well drilling fluids and foodstuifs in particular. This invention also relates to a process for obtaining the novel polysaccharide in yields of 40-45 percent based on the carbohydrate nutrient rather than in commercially unattractive yields of only about 15-20 percent obtained when the amount of nitrogen source in the fermentation medium is not restricted (see FIG. I) and certain metal ions, namely Mg and Mn are not available.

More particularly this invention tion in commercially acceptable yields of a novel watersoluble uronic acid polysaccharide, the constituent suggenus Arthrobacter, no prior art members of which genus have ever been known to produce a polysaccharide gum.

Still more particularly this invention relates to the dispreviously undescribed microorganisms belonging to the diphtheroidic genus Artho-bacter are capable of producing high yields of a novel watersoluble polysaccharide when fermented under certain aerobic conditions for 3 to 4 days in a medium comprising carbon and restricted nitrogen sources, as well as preferably, trace amounts of manganese in addition to obligatory amounts of magnesium ion.

Certain microbial polysaccharide gums are widely known, e.g., the dextrans which consist entirely of glucose units and are produced from sucrose by the enzymes of Leuconstoc bacteria. Less Well known are the phosphomannan gums produced by yeasts belonging to the genus Hansenula, Anderson et al., Arch. Biochem. Biophys. 89: 289 (1960) whose constituent sugars are mannose and mannose-6-phosphate, the polysaccharide produced by Crypzococcus laurentz'i, containing xylose, mannose, glucuronic acid, and acetyl in the ratio of 1:4:1:1, Cadmus et al., Appl. MicrobioL, 10: 153 (1962), as well as polysaccharide 13-1459 produced by the bacterium Xantlzomonas camp-estrz's, NRRL B-1459, Rogovin et al., J. Biochem. Microbiol. Technol. Eng, 3: 51 (1961) which consists of mannose, glucose, glucu-ronic acid and :acetyl in the ratio of 2:1:1:1.

As already indicated, the constituent sugars of the polysaccharide produced by Arthrobacter viscosus NRRL B-1973 and NRRL B1797 are D-glucose and D-galactose in equimolar proportions, there also being an equimolar proportion of D-mannuronic acid moieties and molar equivalents of O-acetyl groups linked to 5 of the 8 nominal hydroxyl positions of the sugars, thus constituting 25.5 percent of the dry weight of the polysac- 3,314,301 Patented Apr. 18, 1967 charide, see Sloneker et :al., p. 24D, Abstr. of Papers, th Meeting of A.C.S., New York City, Sept. 9, 1963. In other words the 13-1973 polysaccharide comprises highly acetylated repeating units consisting of a mannopyranosyluronic acid residue linked beta 1:3 to a glucopyranosyl residue linked 1:4 to a galactopyranosyl residue which latter is again linked 1:4 to a beginning mannopyranosyluronic acid residue.

One object of our invention is the production of a novel polysaccharide having unique and advantageous properties in solution. Another object is a process for producing a polysaccharide gu-m polymer by fermenting members of novel species of diphtheroidic bacterium found in a soil sample from Guatemala and now definitively identified as belonging to the genus Arthrobacter.

In accordance with standard taxonomic and physiological findings, herein set forth, we have classified the novel polysaccharide-producing organism as belonging to the genus Arthrobacter and have assigned them the species name viscosus as well as the accession numbers NRRL B-1973 and NRRL B-1797.

In accordance with the objects of our invention we have made the discovery that two virtually indistinguishable strains of Arthrobacter, specifically A. viscosus NRRL B-1973 and A. viscosus NRRL B-1797 under certain fermentation conditions elaborate excellent yields of an edible polysacaccharide gum which has a novel constitution and unique characteristics and properties.

Parallel fermentations with inocula of the above organism were made in media comprising enzyme-hydrolyzed casein equivalent to 0.04 percent total nitrogen as the nitrogen source and respectively dextrose, technical maltose,- or pure maltose as the carbon source. Because the yields with technical maltose were 2 to 3 times that obtained with the other carbohydrates, we sought an explanation. Addition to the dextrose-containing medium of the two amino acids known to be present in crude maltose gave no improvement in yield. However, the addition of ash from a sample of technical maltose increased the yield of the polysaccharide by two to three fold, and it was subsequently determined that magnesium was mainly responsible, a level of 0.08 percent MgSO 7H O or molybdenum ions. Maximum yields of polysaccharide having a maximum molecular weight are obtained when the pre-sterillization pH of the medium is 7.0 and 0.4 percent of KH PO 0.8 percent buffer is increased, and on cooling the then increases further. minutes and cooling,

solution, the viscosity On autoclaving at 121 C. for 15 solutions containing about 1.5 percent or more of the polysaccharide actually become gels that tend to retain the shape of the container. presence of KCl, a 0.75 percent solution of the polysaccharide gels on cooling. The addition of 2 percent KCl,

even without heating to solutions containing 0.5 percent or more of the polysaccharide very markedly increases the viscosity. As shown in FIGURE 4 of the drawing, the addition of 0.5 percent of NaCl or CaCl doubles the viscosity, but larger additions of these salts do not result in greater increases. The addition of 0.05 percent aluminum sulfate to a 1.0 percent solution of the polysaccharide is unique in that it causes a 20-fold increase in viscosity, but further additions of aluminum sulfate to beyond the 0.25 percent level result in a precipitous dropping off of viscosity to a level somewhat below that of the original solution. The viscosity is stable to pH variations within the range 5.0-10.0. It is apparent that the above responses to temperature and to salts make the polymer very valuable for a diversity of industrial uses, e.g., as an additive for specialty meats, pudding mixes, etc. and as an important constituent of salt-resistant oil well drilling fluids.

In connection with the following specific example, it should be pointed out that the only difference between fermenting an inoculum of NRRL B-1973 and of NRRL B-1797 is that the former gives a very slightly (ca. 3 percent) greater yield of polymer. Also, shake flasks are not suitable for obtaining suitable yields of the polysaccharide, probably because of poor aeration after the culture fluid begins to thicken. Twenty-liter fermentors having paddles are suitable, the paddles being operated at 200 r.p.m. the first day, 300 rpm. the second day, and 500 r.p.m. the third and fourth days. The yields were significantly reduced when fermeneations were started at 500 r.p.m.

EXAMPLE 1 A paddled 20 l. fermentor containing l. of a sterilized fermentation medium having a pH of 7.0 and the following composition per 100 ml.

Water q.s.a.d. 100.

was inoculated under sterile conditions with 500 ml. of material from a proliferating l-day fermentation of Arthrobacter viscosus NRRL 13-1973 in the same medium and incubated at 25 C. under a positive pressure of 1 atmosphere with air admitted at the rate of 0.25 vol. air/liter per min. The paddles were operated at 200 r.p.m. during the first 24 hours, at 300 r.p.m. during the second 24 hours, and at 500 r.p.m. during the next 48 hours. The viscous culture liquor was then diluted with 4 parts of water plus /2 volume of absolute methanol and supercentrifuged for 30 minutes to precipitate cells and debris. The supernatant containing the polysaccharide polymer was then treated by adding 1 g. potassium acetate per 100 ml. (a like amount of KCl can be substituted for the acetate) and 2.5 vols. of 95 percent ethanol to precipitate 92-96 percent of the polymer therefrom.

The precipitate was collected, redissolved in water, reprecipitated, and lyophilized to obtain 1.4 g. of a dry spongy, white product from each 100 ml. of fermented liquor. Based on the initial carbohydrate nutrient, the yield was 47 percent of a polysaccharide composed of gulcose, galactose, manuronic acid, and acetyl groups, which groups are present in molar proportions of 1:1:1:5.

Tables I and II summarize the taxonomic and physiological characteristics of Arthrobacter viscosus NRRL B-l973 and NRRL B-1797 as compared with the corresponding spectra for known Arthrobacter species.

Strains NRRL B-1797 and B1973 produced extremely viscous growth on a variety of media supplemented with various carbon sources. Stained cells from an 18- hour culture on yeast-malt agar showed the presence of highly plemorophic, gram-negative cells. These cells showed definite changes in morphology from short branched, curved and straight rods at 8-12 hours to staphyococcus-like forms at 24-48 hours. Many of these cells exhibited uneven staining reaction. The cultures failed to initiate growth in synthetic medium with phenol as a carbon source and produced nitrites from nitrates, thus eliminating Mycoplana as a generic possibility. Growth did not occur on nitrogen-free synethtic media, thus eliminating the genus Azotobacter. Growth occurred on mannitol-calcium-glycerophosphate agar of Riker et 211. without browning of media; also these cultures produced large circular colonies on nutrient gelatin. These characteristics eliminate Agrobacterium radiobacter as a generic possibility.

The occasional formation of with uneven staining appeared diphtheroids. The growth on inorganic nitrogen and the gram-negative staining reaction eliminated Corynebacteriurn and pointed toward Arthrobacter as the probable genus.

The isolated cultures were compared physiologically and morphologically with known species of Arthrobacter. All control cultures were grown in media in which a maximum extent of polymer production was shown by B1973 and B-l797. This medium consisted of 0.25 percent enzyme hydrolyzed casein, 3 percent commercial corn sugar, MgSO -7H O, MHSO4'4H2O and dibasic potassium phosphate buffer, pH 7.0. None of the control organisms produced any polysaccharide under these conditions. The differences and similiarities between controls and unknown cultures are summarized in Table I.

The differences noted are sufficient to designate a new species. The name Arthrobacter viscosus, sp. n. is proposed with respect to the formation of Y, T,,? GU57 typical of one of the soil slimes in simple basal medium supplemented with each of 10 different carbon sources. Strain NRRL B1797 is designated as the type species since it was the first one studied. NRRL B-l973, since it closely resembles B1797 in physiology and morphology, is considered a strain of the same species. The description of type species NRRL B-1797 follows.

Colonial characteristics Agar streak: filiform, moderate growth, glistening,

cream-like mucoid. Agar colonies: large (3 rnrri.), circular, smooth, entire, flat, opaque, glistening, ver'y mucoid. Gelatin colonies: circular, raised, entire, opaque, cream, smooth, glistening, very mucoid. Soil extract agar slant: growth abundant, white, filiform, very glistening, mucoid. Asparagine agar slant: growth moderately abundant, white, filiform, glistening, very mucoid. Asparagine colonies: 11.5 mm. circular, translucent, white. Potato: growth moderate, viscous, glistening, filiform, light brown. Mannitol-Ca-glycerophosphate agar slant: very mucoid, white, raised, filiform, glistening. M annitol-Ca-glycerophosphate agar colonies: 4.5 mm., spreading, raised, glistening, very mucoid, white precipitate formed around colonies, no brown precipitate. Yeast-malt slant: (Haynes et al., (1955)): heavy, mucoid, white growth which is moderate in 24 hours and profuse in 48 hours.

Physiology Aerobic, very slightly catalase positive. No acid produced from glucose, sucrose, lactose or mannitol; final pH was 7.7. Siight acid production was noted in glycerol (pH 6.2). No acid production from arabinose (pH 7.9), or from raffinose and Z-methyl-glucoside (pH 8.3). No gas produced from any of the tested carbohydrates. No

duced from cysteine or thiosulfate. Growth occurred on inorganic nitrogen sources supplemented with various carbon sources. No cellulose activity. Optimum temperature, 25 C. to 28 C. Growth greatly reduced at 37 C.

taro: growth moderately abundant, glistening, soft, filiform, cream-butt. Y east-malt slant: viscous, profuse white growth, slightly mucoid in 24 hours. Mannitol-Ca-glyce'rol-phosphate colonies: 3-3.5 mm., raised, glistening, mu-

as well as at C. The optimum pH (FIG. I) was 6.1 5 coid, circular, white precipitate formed around colonies, and growth was sharply reduced at pH values below 5.0 no browning of medium. and above 7.0.

Strain NRRL 13-1797. The optimum temperature for Physlology production of the gum was C. Optimum pH for gum production was 7 with none produced at pH values 10 Aerobic, weakly catalase positive. l Io aCIdPIOdUCtIOII below 4.0 with reduced production at 5.0, 6.0, 8.0, and sucrose, lactose or manmtol- Final P of 9 and none at PH 10 (FIG fermentation of glucose was 7 .7, sucrose and lactose, 7 5, and mannitol, 7.3. Slight acid production Was noted in h I glycerol (pH 6.2). Basic products resulted from fermen- IP 0 Ogy l5 t'ations of arabinose, rafiinose and aamethyl glucoside, Cells used for study were grown in nutrient broth on (PH NO gelatm 'hquefactlon Shown after 3 a rotary shaker at 24 a months lncubatron. Indole not produced. Acetylmethyl- At 12 hours gram-ne ative long and short rods were carbmol not produced Nltptes w produced from common Malay i n and Shaped cells trates. No starch hydrolysis. Litmus milk completely were present as well is curved rbds. Most of these cells 0 Cleared. In one month Wlth no formatmn; reducnop were unevenly stained. Some of the longer rods had began m 25 days complete In 2 days; a hard 176.111- 24 heavily stained granules in a lighter stained cell. The ole g m about 3 a the fl g shorter rods almost approached coccoid forms and had washs 1g aslc' f g g 1 g heavier stained ends and lighter stained center portions. t 6 so e.source 9 2 not uce At 2448 hours staphlococcus-like groupings of cell were 25 elthfer cysteine or thlosu l Growth occuneq on morpredorninant. A few large single coccoids were present. gamlc nitrogen P only Shght polymer produ'ctlon noted Flagella stains were made on cells grown on YM slants on m0r.gamc mtrogen. plus gluctgse; moderate polymer and suspended in distilled H O. Observations were made production. shown on morgamc mtrogen Plus crude at 12 hours. Flagella were not observed, but highly plotoss Opnmum tempefature 25-28 NO growth morphic and forms were again Observed curred at 37 C. and little growth was shown after 34 with this staining procedure days incubation at 10 C. The optimum pH (FIG. 1) Strain NRRL B1973 produced viscous growth in liquid if? f fg greatly reduced at values media, but its growth on yeast-malt slants and other solid e ow an a We media was not extremely mucoid. Since this organism I h l differs in this respect and in one physiological aspect, the Mom 0 production of urease, it will be considered a variant of 8-1797. Following is the description of this culture. At 1249 hours i gram-Hagan? liods were Present Some were evenly stained, but the ma ority were unevenly stained. Some Y, X, U, and T forms were pres- C010 Characterlmcs 40 cut and these also were unevenly stained. The short rods Agar streak" fiiliform raised glistening viscous Agar had hfavily stainerlqhenfis and g lightlyjtainfid in centr-a portion. e onger ro s appeare t-o ave colonies: 3 mm., circular, raised, glistening, viscous, white heavy Staining granules in a lighter Stained celL P Soil fihform moderate After 2448 hours micrococcus-like cell groupings were glistening, soft, viscous, raised, cream-colored. Gel atm present and predominant These cells were unevenly colonies: circular, 2 3 mm., smooth, ralsed, glistenm-g, Stained but appeared to be grammegafive, p q I10 llquefactlon Asplll'aglfw 8 Slant! growth Flagella staining of cells grown on YM slant and susmoderately abundant, light Cr am, shgh ly glistellpended in distilled H O, showed the presence of no flagella, ing, wrinkled edge. Asparagine agar colonies: 1 mm., but the highly pleomorphic Y, X, and U forms were circular, translucent, white, raised, slightly mucoid. P0- plainly visible with this staining technique.

TABLE I.-COMPARISONS BETWEEN KNOWN STRAINS OF ARTHROBACTER AND NEW STRAI NS m a A. pascens A. globz'formis A. simpler A. org dam A. aurescens A. tumescms NRRL B-2880 NRRL 1814 NRRL B 2884 Jensen (1954) Sguros (1955) NRRL 13-2879 NRRL B-2881 Gelatin Lique- +stratiformfaction. Starch hydr01ysis- +weak. HzS Prodnction Slight in cysin cysteine Cysteine thio Chr0mogenesis 0:552: White White Cream Yellow and Paleyell0w Gray, cream.

gray. Catalase Motility None. None. N0ne None. Citrate utilizatiorn. N02 N03 utilized as sole N source. B12, thiamin, or

terrogens factor (Loehhead 1953) needed for growth.

TABLE I.-COHPARISONS BETWEEN KNOWN STRAINS OF ARTHROBACTER AND NEW STRAINS.Contil1ucd A. viscosus A. flavescena Lochhead (1958) NRRL 13-1973 NRRL 13-1797 A. ten-wens Lochhead and Burton (1953) A. duodecadis Lochhcad (1958) A. citreusNRRL at'ocyaneus B4258, 3-2882 83 A NR Gelatin Liquefaction.

Starch hydrolysis- H28 Production Cllronlogellesis.

Gatalase Slight Slight Moti1ity S Feeoly... Feebly. Citrate utilization NO: N03 Utilized as sole N source.

Bu, thiamin, or Terrcgens facterregens ctor tor thia- (Lochhead i953) mine biotin. needed for growth.

TABLE IL-COMPARISONS BETWEEN KNOWN STRAINS OF ARTHROBACTER AND NEW STRAINS A. pascens A. globiformis A. simples: A. ora dam A. aurescem A. iumesvcm NRRL B-2880 Jensen (1954) Sgill'Os (1955) NRRL 13-2879 NRRL 13-2881 NRRL 1814 NRRL B-2884 NO1iromNOa. N2 from N02 Growth viscous in No. No

liquid media. Growth at 57 C Slight.-. Litmus milk Clearing slow Clearing slow. Reduction Slow Clearing rcduc- Clearing alkaline. clearing. tion alkaline. clearing.

A.visc0sus A. terrcqens A. citreus NRRL Lochhead and A. airocyaneus A. duodecadis A. flavcscens 13-1258, B 2882 Burton (1953) NRRL B-2883 Lochhead (1958) Lochhead (1958) NRRL 13-1973 NRRL 13-1797 Growth viscous liquid medla. Growth at 37 C Slight. Litmus milk No change No change Soft curd, clear- No change No change Reduction Reduction lng basic. clearing, clearing,

slightly slightly alkaline. alkaline.

Having fully disclosed our invention, we claim: 45 internally agitating the inoculated medium at 25 C. in A method of increasing by about twenty-fold the visthe presence of air for about 96 hours while periodically cosity of a 1 percent aqueous solution of a polysaccharide increasing the rate of agitation by operating the paddles consisting of glucose, galactose, mannuronic acid and O- at 200 r.p.rn. for the first 24 hours, then at 300 rpm. acetyl moieties that are respectively present in the molar for the second 24 hours, and then at 500 rpm. for the proportions of l:1:1:5 and which polysaccharide is pre- 50 remainder of the fermentation, diluting the viscous mepared by the process comprising inoculating a sterilized dium with dilute aqueous methanol, removing unwanted fermentation :nledium present in a paddle equipped fercells and debris, adding ethanol and a salt selected from mentor and having the following composition per 100 ml. the group consisting of potassium chloride and potassium acetate to precipitate the polysaccharide, and isolating the Commercial com Sugar j precipitated polysaccharide, said method comprising add- Enzyme-hydrolyzed casein g 0.25

mg to said 1 percent aqueous solution of said polysaccha- MgSO, 7H O g 0.08 0 5 v MHSOQAHZO 0.005 n e at least .0 percent but not exceeding 0.25 percent KHzPO g 0 4 by weight of aluminum sulfate. Water 100 60 No references cited,

with a culture of a species of diphtheroidic bacterium selected from the group consisting of Arthrobacter viscosus LOUIS MONACELL Prlma'ry Examine NRRL B-1973 and Artlzv'obacter viscosus NRRL 3-1797, J. M. HUNTER, Assistant Examiner. 

